title: Intermediate closed state for glycine receptor function revealed by cysteine cross-linking.
abstract: Pentameric ligand-gated ion channels (pLGICs) mediate signal transmission by coupling the binding of extracellular ligands to the opening of their ion channel. Agonist binding elicits activation and desensitization of pLGICs, through several conformational states, that are, thus far, incompletely characterized at the structural level. We previously reported for GLIC, a prokaryotic pLGIC, that cross-linking of a pair of cysteines at both sides of the extracellular and transmembrane domain interface stabilizes a locally closed (LC) X-ray structure. Here, we introduced the homologous pair of cysteines on the human α1 glycine receptor. We show by electrophysiology that cysteine cross-linking produces a gain-of-function phenotype characterized by concomitant constitutive openings, increased agonist potency, and equalization of efficacies of full and partial agonists. However, it also produces a reduction of maximal currents at saturating agonist concentrations without change of the unitary channel conductance, an effect reversed by the positive allosteric modulator propofol. The cross-linking thus favors a unique closed state distinct from the resting and longest-lived desensitized states. Fitting the data according to a three-state allosteric model suggests that it could correspond to a LC conformation. Its plausible assignment to a gating intermediate or a fast-desensitized state is discussed. Overall, our data show that relative movement of two loops at the extracellular-transmembrane interface accompanies orthosteric agonist-mediated gating.
authors.affiliation: Channel Receptors Unit, Institut Pasteur, 75015 Paris, France.
authors.forename: Marie S
authors.initials: MS
authors.lastname: Prevost
authors.forename: Gustavo
authors.initials: G
authors.lastname: Moraga-Cid
authors.forename: Catherine
authors.initials: C
authors.lastname: Van Renterghem
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Edelstein
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Changeux
authors.forename: Pierre-Jean
authors.initials: PJ
authors.lastname: Corringer
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects / Q000502:physiology; D018686:Anesthetics, Intravenous / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D018360:Crystallography, X-Ray; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D017136:Ion Transport / Q000502:physiology; D008958:Models, Molecular; D015742:Propofol / Q000737:chemistry / Q000494:pharmacology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018009:Receptors, Glycine / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D018686:Anesthetics, Intravenous; D018009:Receptors, Glycine; D015742:Propofol
keywords: cys-loop receptor; protein conformation; signal transduction